1. Field of the Invention
The present invention generally relates to methods and compositions (e.g., particles and kits) for determining activity of a kinase or kinases and, more specifically, to methods and compositions for determining activity/activities of one or more kinases coupled to particles in a multiplexing process using fluorescence detection.
2. Description of the Related Art
The following description and examples are not admitted to be prior art by virtue of their inclusion in this section.
Protein kinases play an important role in regulating cellular signal transduction within living organisms and readily occur in nature. For example, there are more than 500 protein kinases and over 500,000 human phosphorylation sites in the human genome. A protein kinase can be generally defined as an enzyme catalyzing the transfer of phosphate from adenosine triphosphate (ATP) to an amino acid residue. Abnormal expressions of protein phosphorylation events may be associated with several diseases and malignancies in living organisms, particularly humans. As such, monitoring protein kinase activity may be advantageous for detecting diseases and malignancies and/or identifying therapeutic agents for diseases and malignancies (i.e., therapeutic agents for promoting or inhibiting protein kinase activity within a living organism).
As apparent to one skilled in the art of microarray technology, it is generally advantageous to determine the presence and/or concentration of analytes within chemical and biological assays quickly. In addition or alternatively, it may be advantageous to evaluate multiple analytes simultaneously. The simultaneous evaluation of multiple analytes within a single sample is referred to herein as a multiplexing scheme. Conventional techniques for determining kinase activity are not typically suitable for high throughput screening and/or a multiplexing assay. In particular, many conventional techniques for determining kinase activity utilize radioactive isotopes and rely on liquid chromatography and/or mass spectrometry for analysis and, therefore, are not suitable for rapid examination. In addition, such methods do not continuously monitor kinase activity and, consequently, may not render an accurate determination of kinase activity. Other techniques for determining kinase activity involve expensive and specialized biological reagents such as phosphopeptide-specific antibodies. In general, antibody-based microarrays produce a large number of false positives and negatives due to the unpredictable cross-reactivity of antibodies. Consequently, antibody-based kinase activity techniques are generally not amenable to high throughput screening and/or multiplexing assays. Other approaches for determining kinase activity utilize fluorescent sensors which undergo a conformational change upon phosphorylation. A majority of fluorescent sensors employed in conventional assays, however, demonstrate very modest fluorescence changes on phosphorylation, which limits their applicability.
Accordingly, it would be advantageous to develop new methods, particles, and kits for determining kinase activity within an assay, particularly ones that are suitable for high throughput screening and/or multiplexing.